This invention relates to receptor proteins.
The diverse physiological and developmental effects of thyroid hormone receptor (for example, T3)are mediated by the three hormone-binding isoforms of the T3 receptor: TR.alpha.1, TR.beta.1, and TR.beta.2. The effects of the hormone are the consequences of changes in expression of a wide range of target genes that result from T3 binding to these receptors. While it is unknown how binding of the ligand to the receptor actually causes such changes in gene expression, the basic effects on the rate of transcription are believed to be a consequence of direct or indirect protein-protein contacts between the TRs and components of basic transcriptional apparatus, such as RNA polymerase or associated proteins. In addition, interactions of TRs with other transcription factors are thought to result in a variety of complex combinatorial regulatory effects.
In recent years there has been very rapid progress in unravelling the most basic aspects of the mechanism of T3 action in the control of gene expression (see Brent et al., Ann. Rev. Physiol. 53:17-35, 1991 for recent review). It is now clear that the T3 receptors are transcription factors that belong to a related superfamily of nuclear hormone receptors. This family of proteins interacts not only with diverse ligands but also with a complex array of similar DNA binding sites. Like other DNA binding transcription factors, the TRs function by increasing (or, in some cases, decreasing) the rate of transcription initiation from a linked promoter.
Other details of the mechanisms that cause such alterations remain unclear and are the focus of intense study in a number of systems (see Lewin, Cell 61:1161-1164, 1990; Ptashne, Sci. Am. 260:40-47, 1989; Ptashne, and Gann, Nature 346:329-331, 1990, for reviews). However, two broad themes are evident. The first is that transcription factors in general are frequently modular, composed of distinct domains with separate DNA binding and transcriptional regulatory functions. With TRs, for example, it is apparent that the DNA binding and ligand binding domains are quite separate, and experiments with chimeric receptors make it clear that the T3 dependent activation of gene expression can be transferred to heterologous DNA binding domains (see, e.g., Holloway, Proc. Natl. Acad. Sci. USA 87:8160-8164, 1990; Thompson and Evans, Proc. Natl. Acad. Sci. USA 86:3494-3498, 1989).
A second theme is that the functions of transcription factors are believed to be a consequence of protein-protein interactions with the basic transcriptional apparatus. It is thought that these interactions are mediated by proteins called coactivators or adaptors (see Ptashne and Gann, Nature 346:329-331, 1990). These poorly characterized proteins act as bridges between the transcriptional activation domain that is tethered to the DNA by the transcription factor and the RNA polymerase complex bound at the initiation site. Via unknown mechanisms, this interaction leads to an increase in promoter activity.
Protein-protein contacts are also essential for a surprisingly diverse array of positive and negative interactions between transcription factors. Recent results in several systems indicate that this mechanism leads to complex regulatory networks that allow cross talk between various signalling pathways. In the case of TRs, three such interactions have been described to date. The first is the heterodimeric interaction of TRs with the related RXRs (Bugge et al., EMBO J 11:1409-1418, 1992; Kliewer et al., Nature 355:446-449, 1992; Lied et al., Cell 68:377-395, 1992; Marks et al., EMBO J 11:1419-1435, 1992; Yu et al., Cell 67:1251-1266, 1991; Zhang et al., Nature 355:441-446, 1992). TR/RXR heterodimers show higher DNA binding affinity to thyroid hormone response elements (i.e., T3RE sites) initially characterized as binding TR homodimers (see, e.g., Williams et al., J. Biol. Chem. 266:19636-19644, 1991), but heterodimerization does not appear to alter site specificity.
A second, less direct interaction is reflected in the mutually antagonistic effects of the TRs and the c-jun and c-fos protooncogenes (Desbois et al., Cell 67:731-740, 1991; Zhang et al., Mol. Cell. Biol. 11:6016-6025, 1991). The heterodimeric complex of these two leucine zipper transcription factors is frequently referred to as AP-1, although the jun-jun homodimers and other complexes containing related but less well characterized proteins can also bind the consensus AP-1 site. Such sites are also referred to as TPA response elements (i.e., TREs) (here distinguished from T3REs) because the induction of protein kinase C activity by TPA or other phorbol esters results in a very rapid induction of AP-1 activity (reviewed in (Curran and Franza, Cell 55:395-397, 1988). The activity of the TRs is antagonized by coexpression of active jun or fos, and the TRs exert a complimentary inhibition of jun and fos activity. Although the mechanism of this interaction is unknown, it does not require the presence of overlapping DNA binding sites. Thus, TRs can antagonize TPA response on a promoter that does not contain a T3RE, and jun and fos can antagonize T3 response on a promoter that does not include a TRE. Interestingly, although TRs are always nuclear and are able to bind T3REs whether or not hormone is present, the antagonistic function is only observed when T3 is present.
The antagonistic interaction with jun and fos is also observed with other members of the superfamily, including RARs (Desbois et al., Cell 67:731-740, 1991; Schule et al., Proc. Natl. Acad. Sci. USA 88:6092-6096, 1991) and GRs (Jonat et al., Cell 62:1189-1204; Schule et al., Cell 62:1217-1226, 1990; Yang-Yen et al., Cell 62:1205-1215, 1990). The GR interaction was the first described and has been the best characterized, but the biochemical basis for the effect remains uncertain (see Ponta et al., Acta 1129:255-261, 1992 for a review). Despite the potential importance of this apparent cross-talk between nuclear hormone receptors and the protein kinase C signalling pathway, its physiologic impact also remains unclear.
Finally, TRs have also been reported to interact both functionally and biochemically with the cell-type specific transcriptional activator Pit-1 (Schaufele et al., Mol. Endocrinol. 6:656-665, 1992). In contrast to the antagonistic effects of TRs and AP-1, this interaction apparently leads to synergistic activation.
These distinct mechanisms for the modulation of transcriptional activation remain quite unclear. It is apparent that the identification and characterization of proteins capable of interacting specifically with the TRs could provide important clues to these processes and other potential functions of the receptors, such as regulation of cell proliferation (Halperin et al., Endocrinology 126:2321-2326, 1990). In addition, interacting proteins provide a means of controlling and modulating thyroid hormone receptor function.